Binding assays employing magnetic particles

ABSTRACT

A process is provided for the preparation of magnetic particles to which a wide variety of molecules may be coupled. The magnetic particles can be dispersed in aqueous media without rapid settling and conveniently reclaimed from media with a magnetic field. Preferred particles do not become magnetic after application of a magnetic field and can be redispersed and reused. The magnetic particles are useful in biological systems involving separations.

This is a division of application Ser. No. 493,991 filed May 12, 1983,now U.S. Pat. No. 4,554,488.

TABLE OF CONTENTS

1. Field of the Invention

2. Background of the Invention

2.1. Magnetic Separations in Biological Systems: General Considerations

2.2. Separations in Radioimmunoassays

2.3. Application of Magnetic Separations in Other Biological Systems

3. Nomenclature

4. Summary of the Invention

5. Brief Description of the Figures

6. Detailed Description of the Invention

6.1. Magnetic Particle Preparation

6.2 Silane Coupling Chemistry

6.3. Use of Magnetic Particles in Biological Assays

6.4. Use of Magnetic Particles in Immobilized Enzyme Systems

6.5. Use of Magnetic Particles in Affinity Chromatography

7. Examples

7.1. Preparation of Metal Oxide

7.2. Silanization

7.3. Physical Characteristics of Silanized Magnetic Particles

7.4. Coupling of Aminophenyl Magnetic Particles to Antibodies toThyroxine

7.5. Magnetic Particle Radioimmunoassay for Thyroxine

7.6. Magnetic Particle Radioimmunoassay for Theophylline

7.7. Effect of Variation of Fe²⁺ /Fe³⁺ Ratio of Magnetic Particles on T₄Radioimmunoassay

7.8. Coupling of Carboxylic Acid-Terminated Magnetic Particles to B₁₂Binding Protein

7.8.1. Preparation of Carboxylic Acid-Terminated Magnetic Particles

7.8.2. Carbodiimide Coupling of B₁₂ Binding Protein and Human SerumAlbumin to Carboxylic Acid-Terminated Particles

7.9. Magnetic Particle Competitive Binding Assay for Vitamin B₁₂

7.10. Coupling of Magnetic Particles Coated with 3-Aminopropyl orN-2-Aminoethyl-3-Aminopropyl Silane to Proteins

7.10.1. Coupling of N-2-Aminoethyl-3-Aminopropyl Magnetic Particles toAntibodies to Triiodothyronine

7.10.2. Coupling of N-2-Aminoethyl-3-Aminopropyl Magnetic Particles toAntibodies to Thyroid Stimulating Hormone

7.11. Magnetic Particle Radioimmunoassay for Triiodothyronine

7.12. Magnetic Particle Radioimmunoassay for Thyroid Stimulating Hormone

7.13. Coupling of Magnetic Particles Coated withN-2-Aminoethyl-3-Aminopropyl Silane to Enzymes by Use of Glutaraldehyde

1. FIELD OF THE INVENTION

This invention relates to magnetically responsive particles and to theiruse in systems in which the separation of certain molecules from thesurrounding medium is necessary or desirable. More particularly, theinvention relates to methods for the preparation of magneticallyresponsive particles comprising a metal oxide core surrounded by astable silane coating to which a wide variety of organic and/orbiological molecules may be coupled. The particles (coupled oruncoupled) can be dispersed in aqueous media without rapid gravitationalsettling and conveniently reclaimed from the media with a magneticfield. Preferably, the process provided herein yields particles that aresuperparamagnetic; that is, they do not become permanently magnetizedafter application of a magnetic field. This property permits theparticles to be redispersed without magnetic aggregate formation. Hencethe particles may be reused or recycled. Stability of the silane coatingand the covalent attachent of molecules thereto also contribute toparticle use and reuse.

The magnetically responsive particles of this invention may be coupledto biological or organic molecules with affinity for or the ability toadsorb or which interact with certain other biological or organicmolecules. Particles so coupled may be used in a variety of in vitro orin vivo systems involving separation steps or the directed movement ofcoupled molecules to particular sites, including, but not limited to,immunological assays, other biological assays, biochemical or enzymaticreactions, affinity chromatographic purifications, cell sorting anddiagnostic and therapeutic uses.

2. BACKGROUND OF THE INVENTION 2.1. MAGNETIC SEPARATIONS IN BIOLOGICALSYSTEMS: GENERAL CONSIDERATIONS

The use of magnetic separations in biological systems as an alternativeto gravitational or centrifugal separations has been reviewed [B. L.Hirschbein et al., Chemtech, March 1982: 172-179 (1982); M.Pourfarzaneh, The Ligand Quarterly 5(1): 41-47 (1982); and P. J. Hallingand P. Dunnill, Enzyme Microb. Technol. 2: 2-10 (1980)]. Severaladvantages of using magnetically separable particles as supports forbiological molecules such as enzymes, antibodies and other bioaffinityadsorbents are generally recognized. For instance, when magneticparticles are used as solid phase supports in immobilized enzyme systems[see, e.g., P. J. Robinson et al., Biotech. Bioeng., XV: 603-606(1973)], the enzyme may be selectively recovered from media, includingmedia containing suspended solids, allowing recycling in enzymereactors. When used as solid supports in immunoassays or othercompetitive binding assays, magnetic particles permit homogeneousreaction conditions (which promote optimal binding kinetics andminimally alter analyte-adsorbent equilibrium) and facilitate separationof bound from unbound analyte, compared to centrifugation. Centrifugalseparations are time-consuming, require expensive and energy-consumingequipment and pose radiological, biological and physical hazards.Magnetic separations, on the other hand, are relatively rapid and easy,requiring simple equipment. Finally, the use of non-porousadsorbent-coupled magnetic particles in affinity chromatography systemsallows better mass transfer and results in less fouling than inconventional affinity chromatography systems.

Although the general concept of magnetizing molecules by coupling themto magnetic particles has been discussed and the potential advantages ofusing such particles for biological purposes recognized, the practicaldevelopment of magnetic separations has been hindered by severalcritical properties of magnetic particles developed thus far.

Large magnetic particles (mean diameter in solution greater than 10microns(μ)) can respond to weak magnetic fields and magnetic fieldgradients; however, they tend to settle rapidly, limiting theirusefulness for reactions requiring homogeneous conditions. Largeparticles also have a more limited surface area per weight than smallerparticles, so that less material can be coupled to them. Examples oflarge particles are those of Robinson et al. [supra] which are 50-125μin diameter, those of Mosbach and Anderson [Nature, 270: 259-261 (1977)]which are 60-140μ in diameter and those of Guesdon et al. [J. AllergyClin. Immunol. 61(1): 23-27 (1978)] which are 50-160μ in diameter.Composite particles made by Hersh and Yaverbaum [U.S. Pat. No.3,933,997] comprise ferromagnetic iron oxide (Fe₃ O₄) carrier particles.The iron oxide carrier particles were reported to have diameters between1.5 and 10μ. However, based on the reported settling rate of 5 minutesand coupling capacity of only 12 mg of protein per gram of compositeparticles [L. S. Hersh and S. Yaverbaum, Clin. Chim. Acta, 63: 69-72(1975)], the actual size of the composite particles in solution isexpected to be substantially greater than 10μ.

The Hersh and Yaverbaum ferromagnetic carrier particles of U.S. Pat. No.3,933,997 are silanized with silanes capable of reacting withanti-digoxin antibodies to chemically couple the antibodies to thecarrier particles. Various silane couplings are discussed in U.S. Pat.No. 3,652,761, which is hereby incorporated by reference. That thediameters of the composite particles are probably greater than 10μ maybe explained, at least in part, by the method of silanization employedin the Hersch and Yaverbaum patent. Procedures for silanization known inthe art generally differ from each other in the media chosen for thepolymerization of silane and its deposition on reactive surfaces.Organic solvents such as toluene [H. W. Weetall, in: Methods inEnzymology, K. Mosbach (ed.), 44: 134-148, 140 (1976)], methanol [U.S.Pat. No. 3,933,997] and chloroform [U.S. Pat. No. 3,652,761] have beenused. Silane depositions from aqueous alcohol and aqueous solutions withacid [H. W. Weetall, in: Methods in Enzymology, supra, p. 139 (1976)]have also been used. Each of these silanization procedures employs airand/or oven drying in a dehydration step. When applied to silanizationof magnetic carrier particles such dehydration methods allow thesilanized surfaces of the carrier particles to contact each other,potentially resulting in interparticle bonding, including, e.g.,cross-linking between particles by siloxane formation, van der Waalsinteractions or physical adhesion between adjacent particles. Thisinterparticle bonding yields covalently or physically bonded aggregatesof silanized carrier particles of considerably larger diameter thanindividual carrier particles. Such aggregates have low surface area perunit weight and hence, a low capacity for coupling with molecules suchas antibodies, antigens or enzymes. Such aggregates also havegravitational settling times which are too short for many applications.

Small magnetic particles with a mean diameter in solution less thanabout 0.03μ can be kept in solution by thermal agitation and thereforedo not spontaneously settle. However, the magnetic field and magneticfield gradient required to remove such particles from solution are solarge as to require heavy and bulky magnets for their generation, whichare inconvenient to use in benchtop work. Magnets capable of generatingmagnetic fields in excess of 5000 Oersteds are typically required toseparate magnetic particles of less than 0.03μ in diameter. Anapproximate quantitative relationship between the net force (F) actingon a particle and the magnetic field is given by the equation below(Hirschbein et al., supra):

    F=(X.sub.v -X.sub.v °)VH(dH/dx),

where X_(v) and X_(v) ° are the volume susceptibilities of the particleand the medium, respectively, V is the volume of the particle, H is theapplied magnetic field and dH/dx is the magnetic field gradient. Thisexpression is only an approximation because it ignores particle shapeand particle interactions. Nevertheless, it does indicate that the forceon a magnetic particle is directly proportional to the volume of theparticle.

Magnetic particles of less than 0.03μ are used in so-called ferrofluids,which are described, for example, in U.S. Pat. No. 3,531,413.Ferrofluids have numerous applications, but are impractical forapplications requiring separation of the magnetic particles fromsurrounding media because of the large magnetic fields and magneticfield gradients required to effect the separations.

Ferromagnetic materials in general become permanently magnetized inresponse to magnetic fields. Materials termed "superparamagnetic"experience a force in a magnetic field gradient, but do not becomepermanently magnetized. Crystals of magnetic iron oxides may be eitherferromagnetic or superparamagnetic, depending on the size of thecrystals. Superparamagnetic oxides of iron generally result when thecrystal is less than about 300 Å(0.03μ) in diameter; larger crystalsgenerally have a ferromagnetic character. Following initial exposure toa magnetic field, ferromagnetic particles tend to aggregate because ofmagnetic attraction between the permanently magnetized particles, as hasbeen noted by Robinson et al. [supra] and by Hersh and Yaverbaum[supra].

Dispersible magnetic iron oxide particles reportedly having 300 Ådiameters and surface amine groups were prepared by base precipitationof ferrous chloride and ferric chloride (Fe²⁺ /Fe³⁺ =1) in the presenceof polyethylene imine, according to Rembaum in U.S. Pat. No. 4,267,234.Reportedly, these particles were exposed to a magnetic field three timesduring preparation and were described as redispersible. The magneticparticles were mixed with a glutaraldehyde suspension polymerizationsystem to form magnetic polyglutaraldehyde microspheres with reporteddiameters of 0.1μ. Polyglutaraldehyde microspheres have conjugatedaldehyde groups on the surface which can form bonds to amino-containingmolecules such as proteins. However, in general, only compounds whichare capable of reacting with aldehyde groups can be directly linked tothe surface of polyglutaraldehyde microspheres. Moreover, magneticpolyglutaraldehyde microspheres are not sufficiently stable for certainapplications.

2.2. SEPARATIONS IN RADIOIMMUNOASSAYS

Radioimmunoassay (RIA) is a term used to describe methods for analyzingthe concentrations of substances involving a radioactivity labeledsubstance which binds to an antibody. The amount of radioactivity boundis altered by the presence of an unlabeled test substance capable ofbinding to the same antibody. The unlabeled substance, if present,competes for binding sites with the labeled substance and thus decreasesthe amount of radioactivity bound to the antibody. The decrease in boundradioactivity can be correlated to the concentration of the unlabeledtest substance by means of a standard curve. An essential step of RIA isthe separation of bound and free label which must be accomplished inorder to quantitate the bound fraction.

A variety of conventional separation approaches have been applied toradioimmunoassays (RIA) including coated tubes, particulate systems, anddouble antibody separation methods. Coated tubes, such as described inU.S. Pat. No. 3,646,346, allow separation of bound and free labelwithout centrifugation but suffer from two major disadvantages. First,the surface of the tube limits the amount of antibody that can beemployed in the reaction. Second the antibody is far removed (as much as0.5 cm) from some antigen, slowing the reaction between the antibody andantigen [G. M. Parsons, in: Methods in Enzymology, J. Langone (ed.) 73:225 (1981); and P. N. Nayak, The Ligand Quarterly 4(4): 34 (1981)].

Antibodies have been attached to particulate systems to facilitateseparation [see, e.g., U.S. Pat. Nos. 3,652,761 and 3,555,143]. Suchsystems have large surface areas permitting nearly unlimited amounts ofantibody to be used, but the particulates frequently settle during theassay. The tube frequently must be agitated to achieve even partialhomogeneity [P. M. Jacobs, The Ligand Quarterly 4(4): 23-33 (1981)].Centrifugation is still required to effect complete separation of boundand free label.

Antibodies may react with labeled and unlabeled molecules followed byseparation using a second antibody raised to the first antibody [Id.].The technique, termed the double antibody method, achieves homogeneityof antibody during reaction with label but requires an incubation periodfor reaction of first and second antibodies followed by a centrifugationto pellet the antibodies.

Antibodies have been attached to magnetic supports in an effort toeliminate the centrifugation steps in radioimmunoassays fornortriptyline, methotrexate, digoxin, thyroxine and human placentallactogen [R. S. Kamel et al., Clin. Chem., 25(12): 1997-2002 (1979); R.S. Kamel and J. Gardner, Clin. Chim. Acta, 89: 363-370 (1978); U.S. Pat.No. 3,933,997; C. Dawes and J. Gardner, Clin. Chim. Acta, 86: 353-356(1978); D. S. Ithakissios et al., Clin. Chim. Acta, 84: 69-84 (1978); D.S. Ithakissios and D. O. Kubiatowicz, Clin. Chem. 23(11): 2702-2079(1977); and L. Nye et al., Clin. Chim. Acta, 69: 387-396 (1976),repectively, hereby incorporated by reference]. Such methods suffer fromlarge particle sizes (10-100μ in diameter) and require agitation to keepthe antibody dispersed during the assay. Since substantial separationoccurs from spontaneous settling in the absence of a magnetic fieldthese previous methods are in fact only magnetically assistedgravimetric separations. The problem of settling was addressed by Daviesand Janata whose approach in U.S. Pat. No. 4,177,253 was to employmagnetic particles comprising low density cores of materials such ashollow glass or polypropylene (4-10μ in diameter) with magnetic coatings(2 mμ-10μ thick) covering a proportion of the particle surface.Anti-estradiol antibodies were coupled to such particles and theirpotential usefulness in estradiol RIAs was demonstrated. While thisapproach may have overcome the problem of settling, the particle sizeand the magnetic coating nonetheless present limitations on surface areaand hence limitations on the availability of sites for antibodycoupling.

2.3. APPLICATION OF MAGNETIC SEPARATIONS IN OTHER BIOLOGICAL SYSTEMS

Magnetic separations have been applied in other biological systemsbesides RIA. Several nonisotopic immunoassays, such asfluoroimmunoassays (FIA) and enzyme-immunoassays (EIA) have beendeveloped which employ antibody-coupled (or antigen-coupled) magneticparticles. The principle of competitive binding is the same in FIA andEIA as in RIA except that fluorophores and enzymes, respectively, aresubstituted for radioisotopes as label. By way of illustration, M.Pourfarzaneh et al. and R. S. Kamel et al. developed magnetizablesolid-phase FIAs for cortisol and phenytoin, respectively, utilizingferromagnetic-cellulose/iron oxide particles to which antibodies werecoupled by cyanogen bromide activation [M. Pourfarzaneh et al., Clin.Chem., 26(6): 730-733 (1980); R. S. Kamel et al., Clin. Chem., 26(9):1281-1284 (1980)].

A non-competitive solid phase sandwich technique EIA for the measurementof IgE was described by J.-L. Guesdon et al. [J. Allergy Clin. Immunol.,61(1): 23-27 (1978)]. By this method, anti-IgE antibodies coupled byglutaraldehyde activation to magnetic polyacrylamideagarose beads areincubated with a test sample containing IgE to allow binding. Bound IgEis quantitated by adding a second anti-IgE antibody labeled with eitheralkaline phosphatase or β-galactosidase. The enzyme-labeled secondantibody complexes with IgE bound to the first antibody, forming thesandwich, and the particles are separated magnetically. Enzyme activityassociated with the particles, which is proportional to bound IgE isthen measured permitting IgE quantitation.

A magnetizable solid phase non-immune radioassay for vitamin B₁₂ hasbeen reported by D. S. Ithakissios and D. O. Kubiatowicz [Clin. Chem.23(11): 2072-2079 (1977)]. The principle of competitive binding innon-immune radioassays is the same as in RIA with both assays employingradioisotopic labels. However, while RIA is based on antibody-antigenbinding, non-immune radioassays are based on the binding or interactionof certain biomolecules like vitamin B₁₂ with specific or non-specificbinding, carrier, or receptor proteins. The magnetic particles ofIthakissios and Kubiatowicz were composed of barium ferrite particlesembedded in a water-insoluble protein matrix.

In addition to their use in the solid phase biological assays justdescribed, magnetic particles have been used for a variety of otherbiological purposes. Magnetic particles have been used in cell sortingsystems to isolate select viruses, bacteria and other cells from mixedpopulations [U.S. Pat. Nos. 3,970,518; 4,230,685; and 4,267,234, herebyincorporated by reference]. They have been used in affinitychromatography systems to selectively isolate and purify molecules fromsolution and are particularly advantageous for purifications fromcolloidal suspensions [K. Mosbach and L. Anderson, Nature 170: 259-261(1977), hereby incorporated by reference]. Magnetic particles have alsobeen used as the solid phase support in immobilized enzyme systems.Enzymes coupled to magnetic particles are contacted with substrates fora time sufficient to catalyze the biochemical reaction. Thereafter, theenzyme can be magnetically separated from products and unreactedsubstrate and potentially can be reused. Magnetic particles have beenused as supports for α-chymotrypsin, β-galactosidase [U.S. Pat. No.4,152,210, hereby incorporated by reference] and glucose isomerase [U.S.Pat. No. 4,343,901, hereby incorporated by reference] is immobilizedenzyme systems.

3. NOMENCLATURE

The term "magnetically responsive particle" or "magnetic particle" isdefined as any particle dispersible or suspendable in aqueous mediawithout significant gravitational settling and separable from suspensionby application of a magnetic field, which particle comprises a magneticmetal oxide core generally surrounded by an adsorptively or covalentlybound sheath or coat bearing organic functionalities to whichbioaffinity adsorbents may be covalently coupled. The term"magnetocluster" is a synonym of "magnetically responsive particle" and"magnetic particle".

The term "metal oxide core" is defined as a crystal or group (orcluster) of crystals of a transition metal oxide having ferrospinelstructure and comprising trivalent and divalent cations of the same ordifferent transition metals. By way of illustration, a metal oxide coremay be comprised of a cluster of superparamagnetic crystals of an ironoxide, or a cluster of ferromagnetic crystals of an iron oxide, or mayconsist of a single ferromagnetic crystal of an iron oxide.

The term "bioaffinity adsorbent" is defined as any biological or otherorganic molecule capable of specific or nonspecific binding orinteraction with another biological molecule, which binding orinteraction may be referred to as "ligand/ligate" binding or interactionand is exemplified by, but not limited to, antibody/antigen,antibody/hapten, enzyme/substrate, enzyme/inhibitor, enzyme/cofactor,binding protein/substrate, carrier protein/substrate,lectin/carbohydrate, receptor/hormone, receptor/effector orrepressor/inducer bindings or interactions.

The term "coupled magnetically responsive particle" or "coupled magneticparticle" is defined as any magnetic particle to which one or more typesof bioaffinity adsorbents are coupled by covalent bonds, which covalentbonds may be amide, ester, ether sulfonamide, disulfide, azo or othersuitable organic linkages depending on the functionalities available forbonding on both the coat of the magnetic particle and the bioaffinityadsorbent(s).

The term "silane" refers to any bifunctional organosilane and is definedas in U.S. Pat. No. 3,652,761 as an organofunctional andsilicon-functional silicon compound characterized in that the siliconportion of the molecule has an affinity for inorganic materials whilethe organic portion of the molecule is tailored to combine withorganics. Silanes are suitable coating materials for metal oxide coresby virtue of their silicon-functionalities and can be coupled tobioaffinity adsorbents through their organofunctionalities.

The term "superparamagnetism" is defined as that magnetic behaviorexhibited by iron oxides with crystal size less than about 300 ÅA, whichbehavior is characterized by responsiveness to a magnetic field withoutresultant permanent magnetization.

The term "ferromagnetism" is defined as that magnetic behavior exhibitedby iron oxides with crystal size greater than about 500 Å, whichbehavior is characterized by responsiveness to a magnetic field withresultant permanent magnetization.

The term "ferrofluid" is defined as a liquid comprising a colloidaldispersion of finely divided magnetic particles of subdomain size,usually 50-500 Å, in a carrier liquid and a surfactant material, whichparticles remain substantially uniformly dispersed throughout the liquidcarrier even in the presence of magnetic fields of up to about 5000Oersteds.

The term "immunoassay" is defined as any method for measuring theconcentration or amount of an analyte in a solution based on theimmunological binding or interaction of a polyclonal or monoclonalantibody and an antigen, which method (a) requires a separation of boundfrom unbound analyte; (b) employs a radioisotopic, fluorometric,enzymatic, chemiluminescent or other label as the means for measuringthe bound and/or unbound analyte; and (c) may be described as"competitive" if the amount of bound measurable label is generallyinversely proportional to the amount of analyte originally in solutionor "non-competitive" if the amount of bound measurable label isgenerally directly proportional to the amount of analyte originally insolution. Label may be in the antigen, the antibody, or in doubleantibody methods, the second antibody. Immunoassays are exemplified by,but are not limited to, radioimmunoassays (RIA), immunoradiometricassays (IRMA), fluoroimmunoassays (FIA), enzyme immunoassays (EIA), andsandwich method immunoassays.

The term "binding assay" or "non-immune assay" is defined as any methodfor measuring the concentration or amount of an analyte in solutionbased on the specific or nonspecific binding or interaction, other thanantibody/antigen binding or interaction, of a bioaffinity adsorbent andanother biological or organic molecule, which method (a) requires aseparation of bound from unbound analyte; (b) employs a radioisotopic,fluorometric, enzymatic, chemiluminescent or other label as the meansfor measuring the bound and/or unbound analyte; and (c) may be describedas "competitive" if the amount of bound measurable label is generallyinversely proportional to the amount of analyte originally in solutionor "non-competitive" if the amount of bound measurable label isgenerally directly proportional to the amount of analyte originally insolution.

The term "immobilized enzyme reaction" is defined as any enzymaticallycatalyzed biochemical conversion or synthesis or degradation wherein theenzyme molecule or active site thereof is not freely soluble but isadsorptively or covalently bound to a solid phase support, which supportis suspended in or contacted with the surrounding medium and which maybe reclaimed or separated from said medium.

The term "affinity chromatography" is defined as a method forseparating, isolating, and/or purifying a selected molecule from itssurrounding medium on the basis of its binding or interaction with abioaffinity adsorbent adsorptively or covalently bound to a solid phasesupport, which support is suspended in or contacted with the surroundingmedium and which may be reclaimed or separated from said medium.

4. SUMMARY OF THE INVENTION

This invention provides novel magnetic particles useful in biologicalapplications involving the separation of molecules from or the directedmovement of molecules in the surrounding medium. Methods andcompositions for preparing and using the magnetic particles areprovided.

The magnetic particles comprise a magnetic metal oxide core generallysurrounded by an adsorptively or covalently bound silane coat to which awide variety of bioaffinity adsorbents can be covalently bonded throughselected coupling chemistries. The magnetic metal oxide core preferablyincludes a group of superparamagnetic iron-oxide crystals, the coat ispreferably a silane polymer and the coupling chemistries include, butare not limited to, diazotization, carbodiimide and glutaraldehydecouplings.

The magnetic particles produced by the method described herein canremain dispersed in an aqueous medium for a time sufficient to permitthe particles to be used in a number of assay procedures. The particlesare preferably between about 0.1 and about 1.5μ in diameter. Remarkably,preferred particles of the invention with mean diameters in this rangecan be produced with a surface area as high as about 100 to 150 m² /gm,which provides a high capacity for bioaffinity adsorbent coupling.Magnetic particles of this size range overcome the rapid settlingproblems of larger particles, but obviate the need for large magnets togenerate the magnetic fields and magnetic field gradients required toseparate smaller particles. Magnets used to effect separations of themagnetic particles of this invention need only generate magnetic fieldsbetween about 100 and about 1000 Oersteds. Such fields can be obtainedwith permanent magnets which are preferably smaller than the containerwhich holds the dispersion of magnetic particles and thus, may besuitable for benchtop use. Although ferromagnetic particles may beuseful in certain applications of the invention, particles withsuperparamagnetic behavior are usually preferred since superparamagneticparticles do not exhibit the magnetic aggregation associated withferromagnetic particles and permit redispersion and reuse.

The method for preparing the magnetic particles may compriseprecipitating metal salts in base to form fine magnetic metal oxidecrystals, redispersing and washing the crystals in water and in anelectrolyte. Magnetic separations may be used to collect the crystalsbetween washes if the crystals are superparamagnetic. The crystals maythen be coated with a material capable of adsorptively or covalentlybonding to the metal oxide and bearing organic functionalities forcoupling with bioaffinity adsorbents.

In one embodiment the coating around the metal oxide core is a polymerof silane. The silanization may be performed by redispersing themagnetic metal oxide crystals in an acidic organic solution, adding anorganosilane, dehydrating by heating in the presence of a wetting agentmiscible both in water and the organic solution, and washing theresulting magnetic silanized metal oxides. Alternatively, silanizationmay be performed in acidic aqueous solution.

The magnetic particles of this invention can be covalently bonded byconventional coupling chemistries to bioaffinity adsorbents including,but not limited to, antibodies, antigens and specific binding proteins,which coupled magnetic particles can be used in immunoassays or otherbinding assays for the measurement of analytes in solution. Such assayspreferably comprise mixing a sample containing an unknown concentrationof analyte with a known amount of labeled analyte in the presence ofmagnetic particles coupled to a bioaffinity adsorbent capable of bindingto or interacting with both unlabeled and labeled analyte, allowing thebinding or interaction to occur, magnetically separating the particles,measuring the amount of label associated with the magnetic particlesand/or the amount of label free in solution and correlating the amountof label to a standard curve constructed similarly to determine theconcentration of analyte in the sample.

The magnetic particles of this invention are suitable for use inimmobilized enzyme systems, particularly where enzyme recycling isdesired. Enzymatic reactions are preferably carried out by dispersingenzyme-coupled magnetic particles in a reaction mixture containingsubstrate(s), allowing the enzymatic reaction to occur, magneticallyseparating the enzyme-coupled magnetic particle from the reactionmixture containing product(s) and unreacted substrate(s) and, ifdesired, redispersing the particles in fresh substrate(s) therebyreusing enzyme.

Affinity chromatography separations and cell sorting can be performedusing the magnetic particles of this invention, preferably by dispersingbioaffinity adsorbent-coupled magnetic particles in solutions orsuspensions containing molecules or cells to be isolated and/orpurified, allowing the bioaffinity adsorbent and the desired moleculesor cells to interact, magnetically separating the particles from thesolutions or suspension and recovering the isolated molecules or cellsfrom the magnetic particles.

It is further contemplated that the magnetic particles of this inventioncan be used in in vivo systems for the diagnostic localization of cellsor tissues recognized by the particular bioaffinity adsorbent coupled tothe particle and also for magnetically directed delivery of therapeuticagents coupled to the particles to pathological sites.

The magnetic particles of this invention overcome problems associatedwith the size, surface area, gravitational settling rate and magneticcharacter of previously developed magnetic particles. Gravitationalsettling times in excess of about 1.5 hours can be achieved withmagnetic particles of the invention, where the gravitational settlingtime is defined to be the time for the turbidity of a dispersion ofparticles of the invention in the absence of a magnetic field to fall byfifty percent. Magnetic separation times of less than about ten minutescan be achieved with magnetic particles of the invention by contacting avessel containing a dispersion of the particles with a pole face of apermanent magnet no larger in volume than the volume of the vessel,where the magnetic separation time is defined to be the time for theturbidity of the dispersion to fall by 95 percent. Furthermore, the useof silane as the coating surrounding the metal oxide core of themagnetic particles described herein makes possible the coupling of awide variety of molecules under an equally wide variety of couplingconditions compared to other magnetic particle coatings known in the artwith more limited coupling functionalities.

Preferred magnetically responsive particles of the invention have metaloxide cores comprised of clusters of superparamagnetic crystals,affording efficient separation of the particles in low magnetic fields(100-1000 Oersteds) while maintaining superparamagnetic properties.Aggregation of particles is controlled during particle synthesis toproduce particles which are preferably small enough to avoid substantialgravitational settling over times sufficient to permit dispersions ofthe particles to be used in an intended biological assay or otherapplication. The advantage of having superparamagnetic cores inmagnetically responsive particles is that such particles can berepeatedly exposed to magnetic fields. Because they do not becomepermanently magnetized and therefore do not magnetically aggregate, theparticles can be redispersed and reused. Even after silanization,preferred particles of the invention having cores made up of clusters ofcrystals exhibit a remarkably high surface area per unit weight and agenerally correspondingly high coupling capacity, which indicates thatsuch particles have an open or porous structure.

None of the prior art magnetic particles used in the biological systemsdescribed in Section 2 above have the same composition, size, surfacearea, coupling versatility, settling properties and magnetic behavior asthe magnetic particles of the invention. The magnetic particles of thisinvention are suitable for many of the assays, enzyme immobilization,cell sorting and affinity chromatography procedures reported in theliterature and, in fact, overcome many of the problems associated withparticle settling and reuse experienced in the past with suchprocedures.

5. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphical representation of the change in turbidity (%concentration) of a suspension of magnetic particles in the presence andabsence of a magnetic field as a function of time.

FIG. 2 is a photomicrograph of superparamagnetic particles silanizedwith 3-aminopropyltrimethoxy silane.

6. DETAILED DESCRIPTION OF THE INVENTION 6.1. Magnetic ParticlePreparation

Preferred magnetic particles of the invention may be made in two steps.First, superparamagnetic iron oxides are made by precipitation ofdivalent (Fe²⁺) and trivalent (Fe³⁺) iron salts, e.g., FeCl₂ and FeCl₃,in base. Secondly an organosilane coating is applied to the iron oxide.

The ratio of Fe²⁺ and Fe³⁺ can be varied without substantial changes inthe final product by increasing the amount of Fe²⁺ while maintaining aconstant molar amount of iron. The preferred Fe²⁺ /Fe³⁺ ratio is 2/1 butan Fe²⁺ /Fe³⁺ ratio of 4/1 also works satisfactorily in the procedure ofSection 7.1 (See also Section 7.7). An Fe²⁺ /Fe³⁺ ratio of 1/2 producesmagnetic particles of slightly inferior quality to those resulting fromthe higher Fe²⁺ /Fe³⁺ ratios. This magnetic oxide tends to "bleed" orbecome soluble during the rinsing procedure of Section 7.1 and theparticle size is more heterogeneous than the resulting from Fe²⁺ /Fe³⁺of 2/1 or 4/1. Nevertheless, it can be silanized to yield a usablemagnetic particle as demonstrated in Section 7.7.

Aqueous solutions of the iron salts are mixed in a base such as sodiumhydroxide which results in the formation of a crystalline precipitate ofsuperparamagnetic iron oxide. The precipitate is washed repeatedly withwater by magnetically separating it and redispersing it until a neutralpH is reached. The precipitate is then washed once in an electrolyticsolution, e.g. a sodium chloride solution. The electrolyte wash step isimportant to insure fineness of the iron oxide crystals. Finally theprecipitate is washed with methanol until a residue of 1.0% (V/V) wateris left.

The repeated use of magnetic fields to separate the iron oxide fromsuspension during the washing steps is facilitated bysuperparamagnetism. Regardless of how many times the particles aresubjected to magnetic fields, they never become permanently magnetizedand consequently can be redispersed by mild agitation. Permanentlymagnetized (ferromagnetic) metal oxides cannot be prepared by thiswashing procedure as they tend to magnetically aggregate after exposureto magnetic fields and cannot be homogeneously redispersed.

Other divalent transition metal salts such as magnesium, manganese,cobalt, nickel, zinc and copper salts may be substituted for iron (II)salts in the precipitation procedure to yield magnetic metal oxides. Forexample, the substitution of divalent cobalt chloride (CoCl₂) for FeCl₂in the procedure of Section 7.1 produced ferromagnetic metal oxideparticles. Ferromagnetic metal oxides such as that produced with CoCl₂,may be washed in the absence of magnetic fields by employingconventional techniques of centrifugation or filtration between washingsto avoid magnetizing the particles. As long as the resultingferromagnetic metal oxides are of sufficiently small diameter to remaindispersed in aqueous media, they may also be silanized and coupled tobioaffinity adsorbents for use in systems requiring a single magneticseparation, e.g. certain radioimmunoassays. Ferromagnetism limitsparticle usefulness in those applications requiring redispersion orreuse.

Magnetic metal oxides produced by base precipitation may be coated byany one of several suitable silanes. The silane coupling materials havetwo features: They are able to adsorptively or covalently bind to themetal oxide and are able to form covalent bonds with bioaffinityadsorbents through organofunctionalities.

When silanization is used to coat the metal oxide cores of the magneticparticles of this invention, organosilanes of the general formulaR-Si(OX)₃ may be used wherein (OX)₃ represents a trialkoxy group,typically trimethoxy or triethoxy, and R represents any aryl or alkyl oraralkyl group terminating in aminophenyl, amino, hydroxyl, sulphydryl,aliphatic, hydrophobic or mixed function (amphipathic) or other organicgroup suitable for covalent coupling to a bioaffinity absorbent. Suchorganosilanes include, but are not limited to,p-aminophenyltrimethoxysilane, 3-aminopropyltrimethoxysilane,N-2-aminoethyl-3-aminopropyltrimethoxysilane, triaminofunctional silane(H₂ NCH₂ CH₂ --NH--CH₂ CH₂ --NH--CH₂ CH₂ --CH₂ --Si--(OCH₃)₃,n-dodecyltriethoxysilane and n-hexyltrimethoxysilane. [For otherpossible silane coupling agents see U.S. Pat. No. 3,652,761,incorporated by reference, supra]. Generally, chlorosilanes cannot beemployed unless provision is made to neutralize the hydrochloric acidevolved.

In one embodiment, the silane is deposited on the metal oxide core fromacidic organic solution. The silanization reaction occurs in two steps.First, a trimethoxysilane is placed in an organic solvent, such asmethanol, water and an acid, e.g., phosphorous acid or glacial aceticacid. It condenses to form silane polymers; ##STR1## Secondly, thesepolymers associate with the metal oxide, perhaps by forming a covalentbond with surface OH groups through dehydration: ##STR2## Adsorption ofsilane polymers to the metal oxide is also possible.

An important aspect of the acidic organic silanization procedure of thisinvention is the method of dehydration used to effect the adsorptive orcovalent binding of the silane polymer to the metal oxide. Thisassociation is accomplished by heating the silane polymer and metaloxide in the presence of a wetting agent miscible in both the organicsolvent and water. Glycerol, with a boiling point of about 290° C., is asuitable wetting agent. Heating to about 160°-170° in the presence ofglycerol serves two purposes. It insures the evaporation of water, theorganic solvent (which may be e.g., methanol, ethanol, dioxane, acetoneor other moderately polar solvents) and any excess silane monomer.Moreover, the presence of glycerol prevents the aggregation or clumpingand potential cross-linking, of particles that is an inherent problem ofother silanization techiques known in the art wherein dehydration isbrought about by heating to dryness.

In another embodiment an acidic aqueous silanization procedure is usedto deposit a silane polymer on the surface of the metal oxide core.Here, the metal oxide is suspended in an acidic (pH approximately 4.5)solution of 10% silane monomer. Silanization is achieved by heating forabout two hours at 90°-95° C. Glycerol dehydration is again used.

The presence of silane on iron oxide particles was confirmed by thefollowing observations. First, after treatment with 6N hydrochloricacid, the iron oxide was dissolved and a white, amorphous residue wasleft which is not present if unsilanized iron oxide is similarlydigested. The acid insoluble residue was silane. Secondly, thediazotization method of Section 7.4 permits the attachment of antibodiesto the particles. Diazotization does not promote the attachment ofunsilanized particles. Finally, the attachment of antibody is extremelystable, far more stable than that resulting from the adsorption ofantibodies to metal oxides.

6.2. Silane Coupling Chemistry

An initial consideration for choosing a silane coating and theappropriate chemistry for coupling bioaffinity adsorbents to magneticparticles is the nature of the bioaffinity adsorbent itself, itssusceptibilities to such factors as pH and temperature as well as theavailability of reactive groups on the molecule for coupling. Forinstance, if an antibody is to be coupled to the magnetic particle, thecoupling chemistry should be nondestructive to the immunoglobulinprotein, the covalent linkage should be formed at a site on the proteinmolecule such that the antibody/antigen interaction will not be blockedor hindered, and the resulting linkage should be stable under thecoupling conditions chosen. Similarly, if an enzyme is to be coupled tothe magnetic particle, the coupling chemistry should not denature theenzyme protein and the covalent linkage should be formed at a site onthe molecule other than the active or catalytic site or other sites thatmay interfere with enzyme/substrate or enzyme/cofactor interactions.

A variety of coupling chemistries are known in the art and have beendescribed in U.S. Pat. No. 3,652,761 incorporated by reference, supra.By way of illustration, diazotization can be used to couplep-aminophenyl-terminated silanes to immunoglobulins. Coupling ofimmunoglobulins and other proteins to 3-aminopropyl-terminted andN-2-aminoethyl-3-aminopropyl-terminated silanes has been accomplished bythe use of glutaraldehyde. The procedure consists of two basic steps:(1) activation of the particle by reaction with glutaraldehyde followedby removal of unreacted glutaraldehyde and (2) reaction of the proteinswith the activated particles followed by removal of the unreactedproteins. The procedure is widely used for the immobilization ofproteins and cells [A. M. Klibanov, Science, 219:722 (1983), herebyincorporated by reference]. If the magnetic particles are coated bycarboxy-terminated silanes, bioaffinity adsorbents such as proteins andimmunoglobulins can be coupled to them by first treating the particleswith 3-(3-dimethylaminopropyl) carbodiimide.

Generally, magnetic particles coated with silanes bearing certainorganofunctionalities can be modified to substitute more desirablefunctionalities for those already present on the surface. For example,diazo derivatives can be prepared from 3-aminopropyltriethoxysilane byreaction with p-nitro-benzoic acid, reduction of the nitro group to anamine and then diazotization with nitrous acid. The same silane can beconverted to the isothiocyanoalkylsilane derivative by reaction of theamino-function group with thiophosgene.

To effect coupling to the magnetic particle, an aqueous solution of abioaffinity adsorbent can be contacted with the silane coated particleat or below room temperature. When a protein (or immunoglobulin) is tobe coupled, generally a ratio of 1:10-1:30, mg protein: mg particle isused. Contact periods of between about 3 to 24 hours are usuallysufficient for coupling. During this period, the pH is maintained at avalue that will not denature the bioaffinity adsorbent and which bestsuits the type of linkage being formed, e.g. for azo linkages, a pH of8-9.

It has been observed that after coupling of antibodies to silane coatedmagnetic particles by either the diazotization, carbodiimide, orglutaraldehyde methods described in greater detail in Section 7.5, 7.8and 7.10, respectively, the antibodies remain magnetic even after thefollowing rigorous treatments: 24 hours at 50° C. in phosphate bufferedsaline (PBS), 21 days at 37° C. in PBS, 30 minutes at 23° C. in 1Msodium chloride, and repeated rinses in ethanol or methanol at roomtemperature. Antibodies adsorbed to iron oxides are substantiallydetached by any of these treatments. These results indicate that thesilane is very tightly associated with the metal oxide and that thecoupling of antibody to the particle results from an essentiallyirreversible covalent coupling. The tight association of the silane tothe metal oxide together with the covalent coupling of bioaffinityadsorbents (e.g., antibodies) are features which impart stability ontocoupled magnetic particles, a commercially important attribute.

6.3. Use of Magnetic Particles in Biological Assays

The magnetic particles of this invention may be used in immunoassays andother binding assays as defined in Section 3. The most prevalent typesof assays used for diagnostic and research purposes areradioimmunoassays, fluoroimmunoassays, enzyme-immunoassays, andnon-immune radioassays, based on the principle of competitive binding.Basically, a ligand, such as an antibody or specific binding protein,directed against a ligate, such as an antigen, is saturated with anexcess of labeled ligate (*ligate). [Alternatively, competitive assaysmay be run with labeled liqand and unlabeled ligate. Non-competitiveassays, so-called sandwich assays, are also widely employed.] By themethod of this invention, the ligand is coupled to magnetic particle.Examples of labels are radioisotopes: tritium, ¹⁴ -carbon, ⁵⁷ -cobaltand, preferably, ¹²⁵ -iodine; fluorometric labels: rhodamine orfluorescein isothiocyanate; and enzymes (generally chosen for the easewith which the enzymatic reaction can be measured): alkaline phosphataseor β-D-galactosidase. If nonlabeled ligate is added to ligand along with*ligate, less *ligate will be found in the ligand-ligate complex as theratio of unlabeled to labeled liqate increases. If the ligand-*ligatecomplex can be physically separated from *ligate, the amount ofunlabeled ligate in a test substance can be determined.

To measure unlabeled ligate, a standard curve must be constructed. Thisis done by mixing a fixed amount of ligand and *ligate and adding aknown amount of unlabeled ligate to each. When the reaction is complete,the ligand-*ligate is separated from *ligate. A graph is then made thatrelates the label in the collected ligand-*ligate complex to the amountof added unlabeled ligate. To determine the amount of unlabeled ligatein an experimental sample, an aliquot of the sample is added to the sameligand-*ligate mixture used to obtain the standard curve. Theligand-*ligate complex is collected and the label measured, and themount of unlabeled ligand is read from the standard curve. This ispossible with any sample, no matter how complex, as long as nothinginterferes with the ligand-*ligate interaction. By the method of thisinvention, the ligand-*ligate complex is separated magnetically fromfree *ligate.

This general methodology can be applied in assays for the measurement ofa wide variety of compounds including hormones, pharmacologic agents,vitamins and cofactors, hematological substances, virus antigens,nucleic acids, nucleotides, glycosides and sugars. By way ofillustration, the compounds listed in Table I are all measurable bymagnetic particles immunoassays and binding assays [see D. Freifelder,Physical Biochemistry: Applications to Biochemistry and MolecularBiology, p. 259, W. H. Freeman and Company, San Francisco (1976)].

                  TABLE I                                                         ______________________________________                                        SUBSTANCES MEASURABLE IN                                                      MAGNETIC PARTICLE ASSAYS                                                      ______________________________________                                        Hormones:                                                                     Thyroid hormones Prolactin                                                    (thyroxine, triiodo-                                                                           Thyrocalcitonin                                              thyronine, thyroid                                                                             Parathyroid hormone                                          binding globulin,                                                                              Human chorionic gonadotrophir                                thyroid- stimulating                                                                           Human placental lactogen                                     hormone, thyroglobulin)                                                                        Posterior pituitary peptides                                 Gastrointestinal hormones                                                                      (oxytocin, vasopressin,                                      (glucagon, gastrin,                                                                            neurophysin)                                                 enteroglucagon,  Bradykinin                                                   secretin, pancreozy-                                                          min, vasoactive                                                               intestinal peptide,                                                           gastric inhibitory pep-                                                       tide, motilin, insulin)                                                       Follicle- stimulating hormone                                                                  Cortisol                                                     Leutenizing Hormone                                                                            Corticotrophin                                               Progesterone     Human growth hormone                                         Testosterone                                                                  Estriol                                                                       Estradiol                                                                     Pharmacologic agents:                                                         Digoxin          Tetrahydrocannabinol                                         Theophylline     Barbiturates                                                 Morphine and opiate                                                                            Nicotine and metabolic                                       alkaloids        products                                                     Cardiac glycosides                                                                             Phenothiazines                                               Prostaglandins   Amphetamines                                                 Lysergic acid and derivatives                                                 Vitamins and cofactors:                                                       D, B12, folic acid, cyclic AMP                                                Hematological substances:                                                     Fibrinogen, fibrin,                                                                            Prothrombin                                                  and fibrinopeptides                                                           Plasminogen and plasmin                                                                        Transferrin and ferritin                                     Antihemophilic factor                                                                          Erthropoietin                                                Virus antigens:                                                               Hepatitis antigen                                                                              Polio                                                        Herpes simplex   Rabies                                                       Vaccinia         Q fever                                                      Several Groups A Psittacosis group                                            arboviruses                                                                   Nucleic acids and nucleotides:                                                DNA, RNA, cytosine                                                            derivatives                                                                   ______________________________________                                    

6.4 Use of Magnetic Particles in Immobilized Enzyme Systems

Enzymes may be coupled to the magnetic particles of this invention bythe methods described in Section 6.2. They may be used in immobilizedenzyme systems, particularly in batch reactors or continuous-flowstirred-tank reactors (CSTR), to facilitate separation of enzyme fromproduct after the reaction has occurred and to permit enzyme reuse andrecycle. A method for using enzyme-coupled magnetic particles inbiochemical reactions was described by Dunnill and Lilly in U.S. Pat.No. 4,152,210, incorporated by reference, supra. The magnetic particlesof this invention may be advantageously substituted for those of Dunnilland Lilly to avoid problems of settling and to allow enzyme recycle.Briefly, substrates are contacted with enzyme-coupled magnetic particlesin a reactor under conditions of pH, temperature and substrateconcentration that best promote the reaction. After completion of thereaction the particles are magnetically separated from the bulk liquid(which may be a solution of suspension) from which product can beretrieved free of enzyme. The enzyme-coupled magnetic particles can thenbe reused. Immobilized enzymes (coupled to the non-magnetic supports)have been used in a number of industrially important enzymaticreactions, some of which are listed in Table II. The magnetic particlesof this invention can be substituted for the non-magnetic solid phasespreviously employed which include glass, ceramics, polyacrylamide,DEAE-cellulose, chitin, porous silica, cellulose beads andalumino-silicates.

                  TABLE II                                                        ______________________________________                                        INDUSTRIALLY IMPORTANT IMMOBILIZED                                            ENZYME REACTIONS                                                              Enzyme        Reactant/Product                                                ______________________________________                                        Amylo-glucosidase                                                                           Maltose/Glucose                                                 Glucose Oxidase                                                                             Glucose/gluconic acid                                           Glucoamylase  Starch/glucose, Dextrin/glucose                                 β-Amylase                                                                              Starch/maltose                                                  Invertase     Sucrose/glucose                                                 Glucose isomerase                                                                           Glucose/fructose                                                Lactase       Lactose/glucose                                                 Trypsin       Proteins/amino acids                                            Aminoacylase  N--acetyl-DL-methionine/methionine                              Lysozyme      Lysis of M. lysodeikticus                                       ______________________________________                                    

6.5. Use of Magnetic Particles in Affinity Chromatography

The process of affinity chromatography enables the efficient isolationof molecules by making use of features unique to the molecule: theability to recognize or be recognized with a high degree of selectivityby a bioaffinity adsorbent such as an enzyme or antibody and the abilityto bind or adsorb thereto. The process of affinity chromatography simplyinvolves placing a selective bioaffinity adsorbent or ligand in contactwith a solution containing several kinds of substances including thedesired species, the ligate. The ligate is selectively adsorbed to theligand, which is coupled to an insoluble support or matrix. Thenonbinding species are removed by washing. The ligate is then recoveredby eluting with a specific desorbing agent, e.g. a buffer at a pH orionic strength that will cause detachment of the adsorbed ligate.

By the method of this invention, magnetic particles may be used as theinsoluble support to which the ligand is coupled. The particles may besuspended in batch reactors containing the ligate to be isolated. Theparticles with bound ligate may be separated magnetically from the bulkfluid and washed, with magnetic separations between washes. Finally, theligate can be recovered from the particle by desorption. The magneticparticles of this invention may be used in a variety of affinity systemsexemplified by those listed in Table III.

                  TABLE III                                                       ______________________________________                                        AFFINITY SYSTEMS                                                              Ligand, immobile entity                                                                          Ligate, soluble entity                                     ______________________________________                                        Inhibitor, cofactor, prosthetic                                                                  Enzymes; apoenzymes                                        group, polymeric substrate                                                    Enzyme             Polymeric inhibitors                                       Nucleic acid, single strand                                                                      Nucleic acid, complementary                                                   strand                                                     Hapten; antigen    Antibody                                                   Antibody (IgG)     Proteins; polysaccharides                                  Monosaccharide; polysaccharide                                                                   Lectins; receptors                                         Lectin             Glycoproteins; receptors                                   Small target compounds                                                                           Binding Proteins                                           Binding Protein    Small target compounds                                     ______________________________________                                    

7. EXAMPLES 7.1. Preparation of Metal Oxide

The metal oxide particles were prepared by mixing a solution of iron(II)(Fe²⁺) and iron(III) (Fe³⁺) salts with base as follows: a solution thatis 0.5M ferrous chloride (FeCl₂) and 0.25M ferric chloride (FeCl₃) (200mls) was mixed with 5M sodium hydroxide (NaOH) (200 mls) at 60° C. bypouring both solutions into a 500 ml beaker containing 100 mls ofdistilled water. All steps were performed at room temperature unlessotherewise indicated. The mixture was stirred for 2 minutes during whichtime a black, magnetic precipitate formed. After settling, the volume ofthe settled precipitate was approximately 175 mls. The concentration ofiron oxide in the precipitate was about 60 mg/ml (based on a yield of11.2 gms of iron oxide as determined infra). This is in contrast tocommercially available magnetic iron oxides, such as Pfizer #2228 γFe₂O₃ (Pfizer Minerals, Pigments and Metals Division, New York, NY), thestandard magnetic oxide for recording tapes, which can attainconcentrations of about 700 mg/ml in aqueous slurry. The comparison isincluded to emphasize the fineness of the particles made by this method.Very fine particles are incapable of dense packing and entrain the mostwater. Larger and denser particles, on the other hand, pack densely,excluding the most water.

The precipitate was then washed with water until a pH of 6-8 was reachedas determined by pH paper. The following washing technique was employed:

The particles were suspended in 1.8 liters of water in a 2 liter beakerand collected by magnetic extraction. The beaker was placed on top of aring magnet, 1/2 inch high and 6 inches in diameter, which caused themagnetic particles to settle. The water was poured off without the lossof particles by holding the magnet to the bottom of the beaker whiledecanting. A similar washing technique was employed for all washesthroughout, except that volumes were adjusted as necessary. Typically,three washes were sufficient to achieve neutral pH. The magnetic oxidewas then washed once with 1.0 liter of 0.02M sodium chloride (NaCl) inthe same beaker.

The water was then replaced with methanol, leaving a trace of water tocatalyze hydrolysis of the methoxy silane (see Section 7.2.). This wasaccomplished by aspirating 800 mls of 0.2M NaCl and bringing the totalvolume to 1 liter with methanol. The material was resuspended, andmagnetically extracted; 800 mls of supernatant were removed, and another800 mls of methanol were added. After three additions of methanol, theoxide was ready for silanization in a solution which was approximately1% (V/V) water. A portion of the precipitate was dried at 70° C. for 24hours and weighed; 11.2 grams of magnetic iron oxide were formed.

It is to be noted that throughout this procedure the magnetic iron oxideparticles, because of their superparamagnetic properties, never becamepermanently magnetized despite repeated exposure to magnetic fields.Consequently, only mild agitation was required to resuspend theparticles during the water washings and methanol replacement treatment.

7.2. Silanization

The magnetic iron oxide particles (see Section 7.1.) suspended in 250mls of methanol containing approximately 1% (V/V) water were placed in aVirtis 23 homogenizer (Virtis Company, Inc., Gardiner, NY). Two grams oforthophosphorous acid (Fisher Scientific Co., Pittsburgh, PA) and 10 mlsof p-aminophenyltrimethoxysilane (A-7025, Petrarch Systems, Inc.,Bristol, PA) were added. In an alternative protocol, 5 mls of glacialacetic acid have been substituted for the 2 gms of orthophosphorousacid. The mixture was homogenized at 23,000 rpm for 10 minutes and at9,000 rpm for 120 minutes. The contents were poured into a 500 ml glassbeaker containing 200 mls of glycerol and heated on a hot plate until atemperature of 160°-170° C. was reached. The mixture was allowed to coolto room temperature. Both the heating and cooling steps were performedunder nitrogen with stirring. The glycerol particle slurry (about 200mls in volume) was poured into 1.5 liters of water in a 2 liter beaker;the particles were washed exhaustively (usually four times) with wateraccording to the technique described in section 7.1.

This silanization procedure was performed with other silanes, including3-aminopropyltrimethoxysilane,N-2-aminoethyl-3-aminopropyltrimethoxysilane, n-dodecyltriethoxysilaneand n-hexyltrimethoxysilane (A-0800, A-0700, D-6224 and H-7334,respectively, Petrarch Systems, Inc., Bristol, PA).

As an alternative to the above silanization procedure, silane has alsobeen deposited on superparamagnetic iron oxide (as prepared in Section7.1) from acidic aqueous solution. Superparamagnetic iron oxide withFe²⁺ /Fe³⁺ ratio of 2 was washed with water as described in Section 7.1.The transfer to methanol was omitted. One gram of particles (about 20mls of settled particles) was mixed with 100 mls of a 10% solution of3-aminopropyltrimethoxysilane in water. The pH was adjusted to 4.5 withglacial acid. The mixture was heated at 90°-95° C. for 2 hours whilemixing with metal stirblade attached to an electric motor. Aftercooling, the particles were washed 3 times with water (100 mls), 3 timeswith methanol (100 mls) and 3 times with water (100 mls), and thepresence of silane on the particles was confirmed.

7.3. Physical Characteristics of Silanized Magnetic Particles

The mean particle diameter was measured by light scattering and thesurface area per gram as measured by nitrogen gas adsorption forp-aminophenyl silanized, 3-aminopropyl silanized, andN-2-aminoethyl-3-aminopropyl silanized particles are summarized in TableIV. The particle surface area is closely related to the capacity of theparticles to bind protein; as much as 300 mg/gm of protein can becoupled to the N-2-aminoethyl-3-aminopropyl silanized particle, farhigher than previously reported values of 12 mg protein/gm of particles[Hersh and Yaverbaum, Clin. Chem. Acta 63: 69 (1975)]. For comparison,the surface areas per gram for two hypothetical spherical particles ofsilanized magnetite are listed in Table IV. The density of thehypothetical particles was taken to be 2.5 gm/cc, an estimate of thedensity of silanized magnetite particles. The diameter of eachhypothetical particle was taken to be the mean diameter of the particlesof the invention next to which entries for the hypothetical particle islisted. Observe that the surface area per gram of the particles of theinvention as measured by nitrogen gas absorption is far greater than thecalculated surface area per gram for perfect spheres of silanizedmagnetite of the same diameter. The greater surface area per gram of theparticles of the invention indicates that the particles of the inventionhave a porous or otherwise open structure. Hypothetical perfect spheresof silanized magnetite having a diameter of 0.01μ have calculatedsurface area per gram of about 120 m² /gm.

                  TABLE IV                                                        ______________________________________                                        CHARACTERISTICS OF                                                            SILANIZED MAGNETIC PARTICLES                                                                          Measured  Hypoth.                                                Mean Diam..sup.1                                                                           Surf. Area.sup.2                                                                        Surf. Area.sup.3                            Silane     (μ)       (m.sup.2 /gm)                                                                           (m.sup.2 /gm)                               ______________________________________                                        N--2 aminoethyl-                                                                         0.561        140       4.3                                         3-aminopropyl                                                                 p-aminophenyl                                                                            0.803        NM.sup.4  --                                          3-aminopropyl                                                                            0.612        122       3.9                                         ______________________________________                                         .sup.1 Diameter (in microns) was measured by light scattering on a Coulte     N--4 Particle Size Analyzer.                                                  .sup.2 Surface area was measured by N.sub.2 gas adsorption.                   .sup.3 Calculated surface area per gram for a perfect sphere with a           density at 2.5 gm/cc.                                                         .sup.4 Not Measured.                                                     

Because the mean diameters of the silanized magnetic particles producedby the procedures of Sections 7.1 and 7.2 are considerably smaller thanthe diameters of other magnetic particles described in the literature,they exhibit slower gravimetric settling times than those previouslyreported. For instance, the settling time of the particles describedherein is approximately 150 minutes in contrast to settling times of:(a) 5 minutes for the particles of Hersh and Yaverbaum [Clin. Chem.,Acta 63: 69 (1975)], estimated to be greater than 10μ in diameter; and(b) less than 1 minute for the particles of Robinson et al. [Biotech.Bioeng. XV:603 (1973)] which are 50-160μ in diameter.

The silanized magnetic particles of this invention are characterized byvery slow rates of gravimetric settling as a result of their size andcomposition; nevertheless they respond promptly to weak magnetic fields.This is depicted in FIG. 1 where the change in turbidity over time of asuspension of silanized magnetic particles resulting from spontaneousparticle settling in the absence of a magnetic field is compared to thechange in the turbidity produced in the presence of a samarium-cobaltmagnet. It can be seen that after 30 minutes the turbidity of thesuspension has changed only slightly more than 10% in the absence of amagnetic field. However, in the presence of a weak magnetic field, theturbidity of the particle suspension drops by more than 95% of itsoriginal value within 6 minutes. In another experiment, a decrease inturbidity of only about 4% in 30 minutes was observed.

A photomicrograph of superparamagnetic particles silanized with3-aminotrimethoxysilanes ("SIN" particles) is shown in FIG. 2. It can beseen that the particles vary in shape and size and that they are made upof a groups or clusters of individual superparamagnetic crystals (lessthan 300 A) which appear roughly spherical in shape.

7.4. Coupling of Aminophenyl Magnetic Particles to Antibodies toThyroxine

First, thyroxine (T₄) antiserum was prepared as follows: 5.0 mls ofserum of sheep immunized with T₄ (obtained from Radioassay SystemsLaboratories, Inc., Carson, CA) were added to a 50 ml centrifuge tube.Two 5.0 ml aliquots of phosphate buffered saline (PBS) were added to thetube followed by 15 mls of 80% saturated ammonium sulfate, pH 7.4, at 4°C. After mixing, the tube was stored at 4° C. for 90 minutes. Themixture was then centrifuged at 3,000 rpm for 30 minutes at 4° C. Thesupernatant fraction was decanted and the pellet resuspended anddissolved to clarity in 5.0 mls of PBS. The T₄ antiserum preparation(1:2 in PBS) was dialyzed against PBS, transferred from the dialysistubing to a 50 ml centrifuge tube to which 40 mls of PBS were added,bringing the total volume to 50 mls. The T₄ antiserum preparation (1:10in PBS) was refrigerated until used for coupling.

To 1740 mg of p-aminophenyl silanized particles in 100 mls of 1Nhydrochloric acid (HCl), 25 mls of 0.6M sodium nitrite (NaNO₂) wereadded. The NaNO₂ was added slowly below the surface of the particle/HClmixture while maintaining the temperature between 0° and 5° C. with caretaken to avoid freezing. After 10 minutes, the mixture was brought to pH7.5-8.5 by addition of 65 mls of 1.2M NaOH and 18 mls of 1M sodiumbicarbonate (NaHCO₃), still maintaining temperature at 0° to 5° C. Then,50 mls of PBS containing 100 mg of the gamma globulin fraction of sheepserum containing antibodies to thyroxine (the T₄ antiserum preparationdescribed supra) were added. The pH was maintained between 7.5-8.5 whilethe mixture was incubated for 18 hours at 0° to 5° C. Theantibody-coupled particles were washed exhaustively with 0.1 M sodiumphosphate buffer, pH 7.2 (3 times), 1M NaCl, methanol, 1M NaCl and 0.1Msodium phosphate buffer again. Wash steps were repeated twice or more.All washes were performed by dispersing the particles and magneticallyseparating them as described in section 7.1. After washing, theparticles were resuspended in PBS and incubated overnight at 50° C. Theparticles were washed in methanol, 1M NaCl and 0.1M sodium phosphatebuffer as before, and twice in Free T₄ Tracer Buffer. The particles wereresuspended in Free T₄ Tracer Buffer and stored at 4° C. until used forradioimmunoassay.

7.5. Magnetic Particle Radioimmunoassay for Thyroxine

The quantity of antibody-coupled magnetic particles to be used in thethyroxine radioimmunoassay (RIA) was determined empirically using thefollowing RIA procedure:

Ten microliters (μls) of standard were pipetted into 12×75 mmpolypropylene tubes followed by 500 μls of tracer and 100 μls ofmagnetic particles. After vortexing, the mixture was incubated at 37° C.for 15 minutes after which time the tubes were placed on a magnetic rackfor 10 minutes. The rack consisted of a test tube holder with acylindrical "button" magnet (Incor 18, Indiana General Magnetic ProductsCorp., Valparaiso, IN) at the bottom of each tube. The magneticparticles with antibody and bound tracer were pulled to the bottom ofthe tubes allowing the unbound tracer to be removed by inverting therack and pouring off supernatants. Radioactivity in the pellet wasdetermined on a Tracor 1290 Gamma Counter (Tracor Analytic, Inc., ElkGrove Village, IL).

The reagents used in the assay were as follows:

Standards were prepared by adding T₄ to T₄ -free human serum. T₄ wasremoved from the serum by incubation of serum with activated charcoalfollowed by filtration to remove the charcoal according to the method ofCarter [Clin. Chem 24, 362 (1978)]. The tracer was ¹²⁵ I-thyroxinepurchased from Cambridge Medical Diagnostics (#155) and was diluted into0.01M Tris buffer containing 100 μg/ml bovine serum albumin, 10 μg/mlsalicylate, 50 μg/ml 8-amilinonaphthalene-8-sulfonic acid at pH 7.4.Magnetic particles at various concentrations in phosphate bufferedsaline (PBS) with 0.1% bovine serum albumin were used in the RIA todetermine a suitable concentration of particles for T₄ measurements. Aquantity of magnetic particles of approximately 50 μg per tube waschosen for the RIA. This amount permitted good displacement of tracerfrom the antibody for the desired concentration range of T₄ (0-32μg/dl).

Having thus determined the optimal quantity, the RIA procedure describedsupra was performed using approximately 50 μg per tube of magneticparticles to construct a radioimmunoassay standard curve for T₄. Thedata obtained from the RIA is presented in Table V.

                  TABLE V                                                         ______________________________________                                        RIA STANDARD CURVE FOR T.sub.4                                                T4 Concentration                                                                            cpm (average of 2 tubes)                                        ______________________________________                                        0 μg/dl    36763                                                           2 μg/dl    24880                                                           4 μg/dl    18916                                                           8 μg/dl    13737                                                           16 μg/dl   10159                                                           32 μg/dl    7632                                                           Total         69219                                                           ______________________________________                                    

7.6. Magnetic Particle Radioimmunoassay for Theophylline

Rabbit anti-theophylline antibodies were prepared and coupled top-aminophenyl silanized particles according to methods similar to thosedescribed in Section 7.4. The anti-theophylline antibody-coupledmagnetic particles were used in a radioimmunoassay with the followingprotocol: 20 μls of theophylline standard (obtained by addingtheophylline to theophylline-free human serum), 100 μls of ¹²⁵I-theophylline tracer (obtained from Clinical Assays, Cambridge, MA),and 1 ml of antibody-coupled magnetic particles were vortexed. After a15 minute incubation at room temperature, a 10 minute magneticseparation was employed. A standard curve was constructed and the dataobtained are shown in Table VI.

                  TABLE VI                                                        ______________________________________                                        RIA STANDARD CURVE FOR THEOPHYLLINE                                           Theophylline Concentration                                                                      cpm (average of 2 tubes)                                    ______________________________________                                        0 μg/dl        35061                                                       2 μg/dl        28217                                                       8 μg/dl        19797                                                       20 μg/dl       13352                                                       60 μg/dl        8148                                                       Total             52461                                                       ______________________________________                                    

7.7. Effect of Variation of Fe²⁺ /Fe³⁺ Ratio of Magnetic Particles on T4Radioimmunoassay

Magnetic iron oxides were made according to the crystallizationprocedure of Section 7.1 by maintaining constant molar amounts of ironbut varying the Fe²⁺ /Fe³⁺ ratio from 4 to 0.5. These particles weresilanized, coupled to anti-T₄ antibodies and used in the T₄ RIA, as inSections 7.2, 7.4 and 7.5, respectively. The variation of Fe²⁺ /Fe³⁺ratio did not substantially affect the performance of these magneticparticles in the T₄ RIA as shown in Table VII.

                  TABLE VII                                                       ______________________________________                                        T.sub.4 RIA STANDARD CURVES USING MAGNETIC                                    PARTICLES WITH VARIED Fe.sup.2+ /Fe.sup.3+ RATIOS                                         cpm (average of 2 tubes)                                          T4 Concentration                                                                            Fe.sup.2+ /Fe.sup.3+ = 4                                                                  Fe.sup.2+ /Fe.sup.3+ = 0.5                          ______________________________________                                        0 μg/dl    35633       35642                                               1 μg/dl    31681       33139                                               2 μg/dl    30572       30195                                               4 μg/dl    24702       25543                                               8 μg/dl    18680       19720                                               16 μg/dl   12803       11625                                               32 μg/dl   10012        8005                                               Total         77866       75636                                               ______________________________________                                    

7.8. Coupling of Carboxylic Acid-Terminated Magnetic Particles to B₁₂Binding Protein

7.8.1. Preparation of Carboxylic Acid-Terminated Magnetic Particles

A superparamagnetic iron oxide was made by the procedure described inSection 7.1 and silanized as in Section 7.2 with3-aminopropyltrimethoxysilane instead of the aminophenyl silane. Theamino group of the silane was then reacted with glutaric anhydride toconvert the termination from an amine to carboxylic acid. The conversionof the termination was accomplished as follows: five grams ofaminopropyl silanized particles in water were washed four times with 1.5liters of 0.1M NaHCO₃ using the washing procedure of Section 7.1. Thevolume was adjusted to 100 mls and 2.85 gm glutaric anhydride was added.The particles were washed two times and the reaction with glutaricanhydride was repeated. The carboxylic acid-terminated magneticparticles were washed five times with water to prepare them for reactionwith protein.

7.8.2. Carbodiimide Coupling of B₁₂ Binding Protein and Human SerumAlbumin to Carboxylic Acid-Terminated Magnetic Particles

To 50 mg of carboxy-terminated magnetic particles in 1 ml of water wereadded 4 mg of 3-(3 dimethylaminopropyl)-carbodiimide. After mixing byshaking for 2 minutes, 0.05 mg of B₁₂ binding protein (intrinsic factor(IF) from hog gut obtained from Dr. R. H. Allen, Denver, CO) and 0.75 mgof human serum albumin (HSA, obtained from Sigma Chemical Co., A-8763)were added to 0.30 ml in water. The pH was adjusted to pH 5.6 andmaintained by the addition of 0.1N HCl or 0.1N NaOH for three hours. Theparticles were then washed with 10 mls of 0.1M Borate with 0.5M NaCl pH8.3, 10 mls of phosphate buffered saline (PBS) with 0.1% HSA, and 10 mlsof distilled water employing the magnetic separation technique as inSection 7.1. Particles were washed three times with PBS and stored inPBS until use.

7.9. Magnetic Particle Competitive Binding Assay for Vitamin B₁₂

Using the IF- and HSA- coupled magnetic particles made by the method ofSection 7.7, a titering of the particles was performed to ascertain thequantity of particles needed in a competitive binding assay for vitaminB₁₂ (B₁₂). The following assay protocol was used:

100 μls of standard and 1000 μls of tracer buffer were added to 12×75 mmpolypropylene tubes. The mixtures were placed into a boiling water bathfor 15 minutes to effect denaturation of binding proteins in human serumsamples. Then 100 μls of various concentrations of magnetic particles inphosphate buffer were added to determine the optimal quantity ofparticles for assaying B₁₂ concentrations between 0 and 2000 picogram/ml(pg/ml). After incubation of the mixtues for 1 hour at room temperature,a magnetic separation of bound and free B₁₂ was performed according tothe procedure of and using the magnetic rack described in Section 7.5.Radioactivity in the pellets was then counted on a Tracor 1290 GammaCounter (Tracor Analytic, Inc., Elk Grove Village, IL).

The reagents used in the assay were as follows: B₁₂ standards wereobtained from Corning Medical and Scientific, Division of Corning GlassWorks, Medfield, MA #474267. They are made with B₁₂ -free human serumalbumin in PBS and sodium azide added as a preservative. The tracer was⁵⁷ Co-B₁₂ (vitamin B₁₂ tagged with radioactive cobalt) from CorningMedical and Scientific, Division of Corning Glass Works, Medfield, MA,#474287. The tracer is in a borate buffer pH 9.2, containing 0.001%potassium cyanide and sodium azide. Magnetic particles were diluted inPBS at various concentrations to determine the quantity of particlesneeded to measure B₁₂ concentrations between 0 and 2000 pg/ml.

A quantity of magnetic particles of approximately 50 μg/tube wasselected and was used in the B₁₂ competitive binding assay supra toconstruct a standard curve; the data are presented in Table VIII.

                  TABLE VIII                                                      ______________________________________                                        B.sub.12 COMPETITIVE BINDING ASSAY STANDARD CURVE                             B.sub.12 Concentration                                                                      cpm (average of 2 tubes)                                        ______________________________________                                         0 pg/ml      5523                                                            100 pg/ml     5220                                                            250 pg/ml     4169                                                            500 pg/ml     3295                                                            1000 pg/ml    2278                                                            2000 pg/ml    1745                                                            Total         16515                                                           ______________________________________                                    

7.10. Coupling of Magnetic Particles Coated withAminoethyl-3-Aminopropyl Silane to Proteins

7.10.1. Coupling of N-2-Aminoethyl-2-Aminopropyl Magnetic Particles toAntibodies to Triiodothyronine

Six-tenths of a gram of N-2-aminoethyl-3-aminopropyl magnetic particles(abbreviated "DIN" particles for "dinitrogen", signifying that theparticles have a N/Si ratio of 2) prepared as in Section 7.2. wereresuspended in water. The particles were washed once in water and thentwice with 30 mls of 0.1M phosphate buffer, pH 7.4 with magneticseparations between washings. After suspending the washed particles in15 mls of 0.1M phosphate, 15 mls of a 5% (V/V) solution ofglutaraldehyde, formed by diluting 25% glutaraldehyde (G-5882, SigmaChemical Co., St. Louis, MO) with 0.1M phosphate, were added. Theparticles were mixed for 3 hours at room temperature by gently rotatingthe reaction vessel. Unreacted glutaraldehyde was washed away with 5additions of 30 mls of 0.1M phosphate buffer. The glutaraldehydeactivated particles were then resuspended in 15 mls of 0.1M phosphate.

Triiodothyronine (T₃) antiserum (1.6 mls, obtained by immunizing rabbitswith T₃ -BSA conjugates) was added to the activated particles andstirred on a wheel mixer at room temperature for 16 to 24 hours. The T₃antibody-coupled particles were washed once with 30 mls of 0.1Mphosphate and suspended in 15 mls of 0.2M glycine solution in order toreact any unreacted aldehyde groups. The suspension was mixed by shakingfor 25 minutes. The antibody-coupled particles were washed with 30 mlsof 0.1 phosphate, 30 mls of ethanol and twice with 150 mls of PBS with0.1% bovine serum albumin (BSA). They were resuspended in PBS, 1% BSAand stored at 4° C. until used for RIA for T₃.

7.10.2. Coupling of N-2-Aminoethyl-3-Aminopropyl Magnetic Particles toAntibodies to Thyroid Stimulating Hormone

The coupling procedure of Section 6.10.1 was followed with minormodifications. Twenty grams of DIN particles were washed three timeswith 1.5 liters of methanol prior to glutaraldehyde activation.Glutaraldehyde activation was performed as in Section 7.10.1. withadjustments for scale.

A goat gamma globulin fraction containing antibodies to human thyroidstimulating hormone (TSH) was coupled to the DIN particles rather thanwhole antisera. Fractionation was accomplished by precipitation ofgammaglobulins with 40% ammonium sulfate followed by dialysis againstPBS. Approximately 4 grams of protein (200 mls at 20 mg/ml) werecoupled. Complete attachment of protein was evident by the absence ofoptical density at 280 nm in the supernatant after coupling. Thisindicated the attachment of about 20 mg of protein per gram ofparticles. The particles were then washed three times with 1.5 liters of1M NaCl, three times with PBS and incubated at 50° C. overnight.Particles were then washed 3 more times in PBS/BSA and titered for usein the TSH assay.

7.11. Magnetic Particle Radioimmunoassay for Triiodothyronine

The quantity of particles to be used in the T₃ RIA was determined in thefollowing assay:

Standards were prepared by adding T₃ to T₃ -free human serum as with T₄(see Section 7.5.)

Tracer was ¹²⁵ IT₃ from Corning Medical and Scientific, Division ofCorning Glass Works, Medfield, MA (#47106).

Magnetic particles were diluted to various concentrations in PBS-BSA todetermine the quantity of particles needed.

The assay protocol was as follows: 50 μls of standard, 100 μls of tracerand 800 μls of DIN magnetic particles were pipetted into 12×75 mmpolypropylene tubes. After vortexing, the tubes were incubated for 2hours at room temperature. The assay was terminated by magneticseparation. By titering the quantity of particles in the assay with a 0ng/ml standard, a quantity of 30 μg/tube was deemed to be optimal forthe assay protocol. Table IX shows the T₃ RIA standard curve dataobtained with these particles.

                  TABLE IX                                                        ______________________________________                                        RIA STANDARD CURVE FOR T.sub.3                                                T.sub.3 Concentration                                                                       cpm (average of 2 tubes)                                        ______________________________________                                        0.0  ng/ml    17278                                                           0.25 ng/ml    15034                                                           0.50 ng/ml    13456                                                           1.00 ng/ml    12127                                                           2.00 ng/ml     8758                                                           4.00 ng/ml     5776                                                           8.00 ng/ml     3897                                                           Total         26946                                                           ______________________________________                                    

7.12. Magnetic Particle Radioimmunoassay for Thyroid Stimulating Hormone

The quantity of particles to be used in the TSH RIA was determined inthe following assay:

Standards were in normal human serum (Corning Medical and Scientific,#47186, Medfield, MA).

Tracer was ¹²⁵ I-rabbit anti-TSH antibody in PBS (Corning Medical andScientific, #474185, Medfield, MA).

Magnetic particles were diluted to various concentrations in PBS-BSA todetermine the quantity of particles needed.

The assay protocol was as follows: 100 μls of standard and 100 μls oftracer were pipetted into 12×75 mm polypropylene tubes, vortexed, andincubated for 3 hours at room temperature. Magnetic particles (500 μls)were added and the mixture was vortexed and incubated for 1 hour at roomtemperature. 500 μls of water were added and the usual magneticseparation was employed to separate bound from unbound tracer. In thepresence of TSH, a sandwich is formed between magnetic antibody (goatanti-TSH antibody, see Section 7.10.1.) TSH and tracer ¹²⁵ I-antibody(rabbit anti-TSH antibody). Thus, increasing concentrations of analyte(TSH) increase the amount of bound radioactivity. Table X shows the TSHRIA standard curve data obtained by this procedure.

                  TABLE X                                                         ______________________________________                                        RIA STANDARD CURVE FOR TSH                                                    TSH Concentration cpm                                                         ______________________________________                                        0           μIU/ml*                                                                              1615                                                    1.5         μIU/ml*                                                                              2309                                                    3.0         μIU/ml*                                                                              3014                                                    6.0         μIU/ml*                                                                              4448                                                    15.0        μIU/ml*                                                                              7793                                                    30.0        μIU/ml*                                                                              11063                                                   60.0        μIU/ml*                                                                              15030                                                   Total             45168                                                       ______________________________________                                         *μIU = micro International Units                                      

7.13. Coupling of Magnetic Particles Coated withN-2-Aminoethyl-3-Aminopropyl Silane to Enzymes by Use of Glutaraldehyde

Magnetic particles (1 gm) were activated with glutaraldehyde as inSection 7.10.1. After washing, the particles were resuspended in 15 mlsof PBS. Then 3 mls of particles (2 gm) were mixed with 5 mg of alkalinephosphates (Sigma Chemical Company, P-9761) or 5 mg of β-glactosidase(Sigma Chemical Company, 5635) dissolved in 2.0 mls of PBS. The coupledparticles were washed with glycine and then washed 5 times with PBS andresuspended in PBS with 0.1% BSA.

Enzyme assays for magnetic alkaline phosphatase activity was performedas follows:

To a 3 ml cuvette 3 mls of 0.05M Tris-HCl were added, pH 8.0, with 3 mMp-nitrophenyl-phosphate. Then 100 μls of diluted magnetic particles withcoupled alkaline phosphatase were added. The increase in optical densityat 410 nm was recorded.

Enzyme assay for magnetic β-galactosidase activity was performed asfollows:

To a 3 ml cuvette 3 mls of 0.1M phosphate were added, pH 7.4, with 0.01Mmercaptoethanol and 0.005M O-nitrophenyl-β-O-galactopyranoside. Then 100μls of diluted magnetic particles coupled to β-galactosidase were added.The increase in optical density at 410 nm was recorded.

It is apparent that many modifications and variations of this inventionas hereinabove set forth may be made without departing from the spiritand scope thereof. The specific embodiments described are given by wayof example only and the invention is limited only by the terms of theappended claims.

We claim:
 1. A method for determining the concentration of a ligate in asolution which comprises:(a) reacting the solution, a known amount oflabeled ligate, and magnetically-responsive particles to which a ligandspecific for the ligate in solution is covalently coupled, to formligand/ligate complexes; (b) magnetically separating themagnetically-responsive particles from the reaction solution; (c)measuring the amount of label associated with themagnetically-responsive particles or remaining free in solution; and (d)relating the amount of label measured in step (c) to a standard curve todetermine ligate concentration,wherein the magnetically-responsiveparticles of step (a) individually comprise a magnetic metal oxide coregenerally surrounded by a coat of polymeric silane, a mass of theparticles being dispersable in aqueous media to form an aqueousdispersion having (i) a fifty-percent-turbidity-decrease settling timeof greater than about 1.5 hours in the absence of a magnetic field, and(ii) a ninty-five-percent-turbidity-decrease separation time of lessthan about 10 minutes in the presence of a magnetic field, the magneticfield being applied to the aqueous dispersion by bringing a vesselcontaining a volume of the dispersion into contact with a pole face of apermanent magnet, the permanent magnet having a volume which is lessthan the volume of the aqueous dispersion in the vessel.
 2. A method fordetermining the concentration of a ligate in a solution whichcomprises:(a) reacting the solution, a known amount of labeled ligate,and magnetically-responsive particles to which a ligand specific for theligate in solution is covalently coupled, to form ligand/ligatecomplexes; (b) magnetically separating the magnetically-responsiveparticles from the reaction solution; (c) measuring the amount of labelassociated with the magnetically-responsive particles or remaining freein solution; and (d) relating the amount of label measured in step (c)to a standard curve to determine ligate concentration,wherein themagnetically-responsive particles of step (a) individually comprise asuperparamagnetic iron oxide core generally surrounded by a coat ofpolymer silane, the iron oxide core including a group of crystals ofiron oxide, an individual particle having a mean diameter as measured bylight scattering between about 0.1μ and about 1.5μ and a surface area asmeasured by nitrogen gas adsorption of at least about 100 m² /gm, a massof the particles being dispersable in aqueous media to form an aqueousdispersion having (i) a fifty-percent-turbidity-decrease settling timeof greater than about 1.5 hours in the absence of a magnetic field, and(ii) a ninety-five-percent-turbidity-decrease separation time of lessthan about 10 minutes in the presence of a magnetic field, the magneticfield being applied to the aqueous dispersion by bringing a vesselcontaining a volume of the dispersion into contact with a pole face of apermanent magnet, the permanent magnet having a volume which is lessthan the volume of the aqueous dispersion in the vessel.
 3. A method fordetermining the concentration of a ligate in a solution whichcomprises:(a) reacting the solution, a known amount of labeled ligate,and magnetically-responsive particles to which a ligand specific for theligate in solution is covalently coupled, to form ligand/ligatecomplexes; (b) magnetically separating the magnetically-responsiveparticles from the reaction solution; (c) measuring the amount of labelassociated with the magnetically-responsive particles or remaining freein solution; and (d) relating the amount of label measured in step (c)to a standard curve to determine ligate concentration,wherein themagnetically-responsive particles of step (a) individually comprise aferromagnetic metal oxide core generally surrounded by a coat ofpolymeric silane, the metal oxide core including a group of crystals ofmetal oxide, an individual particle having a mean diameter as measuredby light scattering between about 0.1μ and about 1.5μ and a surface areaas measured by nitrogen gas adsorption of at least about 100 m² /gm, amass of the particles being dispersable in aqueous media to form anaqueous dispersion having (i) a fifty-percent-turbidity-decreasesettling time of greater than about 1.5 hours in the absence of amagnetic field, and (ii) a ninety-five-percent-turbidity-decreaseseparation time of less than about 10 minutes in the presence of amagnetic field, the magnetic field being applied to the aqueousdispersion by bringing a vessel containing a volume of the dispersioninto contact with a pole face of a permanent magnet, the permanentmagnet having a volume which is less than the volume of the aqueousdispersion in the vessel.
 4. The method of claim 1, 2 or 3 wherein theligand is an antibody.
 5. The method of claim 1, 2 or 3 wherein theantibody is selected from the group consisting of anti-thyroxine,anti-triiodothyronine, anti-thyroid stimulating hormone, anti-thyroidbinding globulin, anti-thyroglobulin, anti-digoxin, anti-cortisol,anti-insulin, anti-theophylline, anti-vitamin B₁₂, anti-folate,anti-ferritin, anti-human chorionic gonadotropin, anti-folliclestimulating hormone, anti-progesterone, anti-testosterone, anti-estriol,anti-estradiol, anti-prolactin, anti-human placental lactogen,anti-gastrin and anti-human growth hormone antibodies.
 6. The method ofclaim 1, 2 or 3 wherein the ligate is selected from the group consistingof hormones, peptides, pharmacological agents, vitamins, cofactors,hematological substances, virus antigens, nucleic acids and nucleotides.7. The method of claim 2 or 3 wherein the ligate is thyroxine and theligand is an anti-thyroxine antibody.
 8. The method of claim 2 or 3wherein the ligate is theophylline and the ligand is ananti-theophylline antibody.
 9. The method of claim 2 or 3 wherein theligate is vitamin B₁₂ and the ligand is vitamin B₁₂ binding protein. 10.The method of claim 2 or 3 wherein the ligate is triiodo-thyronine andthe ligand is an anti-triiodothyronine antibody.
 11. The method of claim2 or 3 wherein the ligate is a thyroid stimulating hormone and theligand is an anti-thyroid stimulating hormone antibody.